Method and compositions for treatment of fungal nail disease

ABSTRACT

Compositions for the treatment of fungal nail disease (onychomycosis), including camphor, menthol, eucalyptus and thymol are described. The ingredients are natural and very effective in treating the nail fungus.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to application Ser. No. 60/118,974,filed Feb. 8, 1999.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to novel topical compositions and methodfor the treatment of fungal nail disease. In particular, the presentinvention relates to the use of GRAS natural plant derivatives for thispurpose.

(2) Description of Related Art

Fungal nail disease (onychomycosis) is the most commonly occurring naildisorder encountered in primary care. Current estimates indicate thatnearly 11 million Americans are affected by fungal nail disease. It canaffect toenails, fingernails or both and is usually caused by infectiousorganisms known as dermatophytes.

Fungal nail disease causes discoloration and thickening of the nail,accumulation of debris under the nail and, in severe cases, detachmentof the nail plate from the nail bed. Toenails are the primary site ofinfection. Fungal nail disease can affect people of any age, gender andrace and may cause discomfort and embarrassment due to the appearance ofthe nails.

Onychomycosis (Tine unguium) is defined as a localized infection of thenail or nail bed primarily caused by a pathogenic fungi; however, yeastsand molds can also cause infection. This disease is not lifethreatening, however, it can cause inconvenience, pain and discomfort tothe individual and serve as a reservoir for infection. Infection canusually be determined by thickened, yellow or brown discolored, friablenail plates (Zaias, N., et al., J. Fam. Pract. 42 513-518 (1996)).

Onychomycosis affects up to 30% of the population by age 60. The mostcommon dermatophytes are Trichophyton rubrum and Trichophytonmentagrophytes (Zaias, N., et al., J. Fam. Pract. 42 513-518 (1996)).Although there are numerous topical agents on the market, none have beenfound to be satisfactory (Zaias & Serrano, Clinical ExperimentalDermatology 14(2) 120-123 (1989)). Consequently, emphasis in researchstudies has focused on oral treatments.

Fungal infection of the fingernails or toenails are caused most commonlyby dermatophytes (Tricophyton rubrum, T. Mentagrophytes, Microsporumcanis, Epidermophyton Floccosum and E. stockdale) which represent about90% of the infection. Yeasts (Candida albicans, C. parapsilosis. and C.krusei) and nondermatophyte molds (Scytalidium hyalinum, S. dimidiatum,Fusarium oxysporum, F. moniliforme, Acremonium chrysogenum, A. strictum,Aspergillus terreus, A. flavus, and Scopulariopsis brevicaulis) areresponsible for 7% and 3% of infections, respectively. These organismsinfect the stratum corneum of the skin, hair and nails (Zaias, N., etal., J. Fam. Pract. 42 513-518 (1996); and Tom, C. M., et al., Am. J.Health-Syst. Pharm. 56 865-871 (1999)).

Immunodeficient patients, such as those with AIDS, the nail infectioncan be severe. Also, diabetics are known to be predisposed to certaincutaneous diseases but their frequency of infection is still not clear(Tom, C. M., et al., Am. J. Health-syst. Pharm. 56 865-871 (1999); andLugo-Somalinos, A., et al., J. Am. Acad. Dermatol. 26 408-410 (1992)).There are four types of onychomycosis: (1) distal subungualonychomycosis (DSO) affecting the nail bed, (2) white superficialonychomycosis (WSO) affecting the surface of the nail plate, (3)proximal subungual onychomycosis (PSO) affecting the ventral andproximal area of the nail plate from the proximal nail fold, and (4)chronic mucocutaneous candidiasis (CMC) affecting the entire thicknessof the nail plate (Zaias, N., et al., J. Fam. Pract. 42 513-518 (1996)).

DSO is the most common form and accounts for about 90% of the cases ofonychomycosis. It is typically a lifelong infection and difficult totreat and is caused mainly by T. rubrum. A traumatized nail can becomeinfected by species of Scytalidium or Scopulariopsis. WSO mainlyinvolves the toenails and manifests itself as small, well-defined whitespots on the surface of the nail plate. It is caused mainly by T.Mentagrophytes. PSO accounts for less than 1% of the cases and is rarein healthy adults but occurs frequently in patients with AIDS. This typeis caused by a preexisting T. rubrum that predates immunosuppression.CMC accounts for less than 1% of onychomycosis cases and invasion of thenail is by C. albicans. Most commonly, the organism originates in theintestinal tract and spreads from the mouth to the hand, eventuallyaffecting the nail plate, nail bed and nail fold (Zaias, N., et al., J.Fam. Pract. 42 513-518 (1996); and Tom, C. M., et al., Am. J.Health-Syst. Pharm. 56 865-871 (1999)).

Onychomycosis never resolves spontaneously and recurrence aftertreatment is common. Treatment is difficult because of the uniqueproperties of the nail unit. Thus, an effective antifungal agent mustenter the affected tissue and persist there in high concentrations. Theexisting therapies must be continuous and used until the infected nailgrows out and even after this lengthy treatment low cure rates and quickrelapse times are common (Zaias, N., et al., J. Fam. Pract. 42 513-518(1996); and Tom, C. M., et al., Am. J. Health-syst. Pharm. 56 865-871(1999)). Treatments with topical creams are not as effective as oraltreatments and prolonged periods of application are required.Griseofulvin, terbinafine and itraconazole are the drugs usually usedfor oral treatment.

Orally administered drugs that are available on the market also sufferfrom various drawbacks. Some are not very effective in controlling thefungal infection, some produce unwanted side effects, and all are quiteexpensive when compared to topical drugs. For example, Einarson, Aridian& Shear (Einarson, et al., British Journal of Dermatology, 130(Supplement 43), 32-34 (1994)) studied the cost-effectiveness ofgriseofulvin (GRI), ketoconazole (KET) and terbinafine (TER). Theexpected treatment costs for toenail onychomycosis were $1049.77 forTER, $1388.54 for GRI and $1936.48 for KET. At the same time, successrates of the treatments were 78.3% for TER, 40.8% for KET and 17.5% forGRI.

A study by Korting, Schafer, Korting, Zienicke, Georgii & Ollert(Korting H., et al., Antimicrobial-Agents-Chemotherapy 37 (10) 2064-2068(1993)) concurs that griseofulvin is minimally successful. In additionto unremarkable success rates and high costs, Zaias & Drachman (Zaias,N., et al., Journal of the American Academy of Dermatology 9(6), 912-919(1983)) also report serious side effects with ketoconazole. Bothgriseofulvin and ketoconazole can cause elevated liver enzymes andketoconazole has been shown to cause anti-androgenic dysfunction inmales and adrenal dysfunction (Zaias & Serrano, Clinical ExperimentalDermatology 14(2), 120-123 (1989)).

Recently itraconazole has been introduced as an effective oral treatmentfor onychomycosis. Its clinical cure rates are between 72% and 80%(Doncker, Decrois, Pierard, Roeland, Woestenborghs, Jacomin, Odds,Heremans, Dockx & Roseeuw, Archives of Dermatology 132(1), 34-41(1996)). However, the cost of itraconazole is high and side effectsinclude elevations of serum transaminases, alkaline phosphatase andbilirubin. Additionally, there are numerous drug interactions, includingphenytoin, rifampin, Carbamazepine, isoniazid, cyclosporin, digoxin,terfenadine and warfarin (Nurses Drug Guide, 1996). Despite these knownproblems, many health care practitioners are currently prescribing pulsetherapy with itraconazole, consisting of monthly one-week cycles of 400mg daily for three to four months (Doncker, Van-Lint, Dockx & Roseeuw,Cutis 56(3), 180-183 (1995)).

In sum, because of the high cost and potentially dangerous side effectsof oral antifungal agents, there are still no solutions for thetreatment of onychomycosis using non-prescription products.Onychomycosis remains a concern for a large number of individuals. Inlight of these circumstances, an alternative topical agent that isinexpensive, and effective is needed.

OBJECTS

It is therefore an object of the present invention to provide topicalcompositions and a method for the use for treating fungal nail diseasewhich produce very significant improvement. Further, it is an object ofthe present invention to provide compositions which use plant materialsfrom natural sources which are GRAS General Recognized As Safe underU.S. regulations. Further still, it is an object of the presentinvention to provide compositions which are inexpensive. These and otherobjects will become increasingly apparent by reference to the followingdescription and the drawings.

SUMMARY OF THE INVENTION

The present invention relates to a method for inhibiting a dermatophytein an infected nail in humans which comprises:

multiple separate applications of an effective amount of a compositionwhich consists of an active ingredient selected from the groupconsisting of camphor, menthol, eucalyptus oil, thy mol and mixturesthereof as active ingredients in a topical carrier to the nail until thedermatophyte is inhibited.

Further the present invention relates to a composition for treating anail infection caused by a dermatophyte which consists essentially of:

(a) an effective amount of an active ingredient selected from the groupconsisting of camphor, menthol, eucalyptus oil, thymol and mixturesthereof; and

(b) a topical carrier, wherein the active ingredient is present in anamount between about 0.01 and 25% by weight of the composition.

Further still, the present invention relates to a kit for the treatmentof a nail infection caused by a dermatophyte which comprises:

(a) a closed openable container containing a composition which consistsessentially of an effective amount of an active ingredient selected fromthe group consisting of camphor, menthol, eucalyptus oil, thymol andmixtures thereof in a topical carrier; and

(b) application means for applying the composition on and under the nailwhich is infected with the dermatophyte.

The compositions can contain between 0.01 and 25% by weight of each ofthe ingredients based upon the weight of the composition. In a mixtureof the four (4) ingredients the amount would be 100%. Preferably theamount is between about 1 and 10% by weight of each of the ingredientsbased upon the weight of the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the age of patients treated bycompositions of the present invention.

FIG. 2 is a bar graph showing the age and diseases of the patients ofFIG. 1.

FIG. 3 is a bar graph showing the results with the patients of FIGS. 1and 2.

FIG. 4 is a bar graph showing the length of treatment of the patients.

FIG. 5 is a chart showing the assessment reading protocol for Example 2.

FIG. 6 is a chart showing the process for extracting a Vapo-rub(Meijer's brand, Michigan) with diethyl ether (VE) or methanol (VM) toremove the solid petroleum carrier.

DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

Antifungal, antimold, and antiyeast assay: Tricophyton rubrum, T.mentagrophytes, Microsporum canis, Epidermophyton floccosum, Scytalidiumhyalinum, S. dimidiatum, Fusarium oxysporum, F. proliferatum, Acremoniumchrysogenum, A. strictum, Aspergillus terreus, A. flavus, andScopulariopsis brevicaulis used for the antifungal and antimold assayswere cultured in Petri dishes containing PDA medium (20 mL). The testorganisms Candida albicans (MSU strain), C. krusei (MSU strain), and C.parapsilosis (MSU strain) used for the antiyeast bioassays were culturedin Petri dishes containing YMG media (20 mL). The cells from a fullygrown plate of each organism were suspended in saline solution (5 mL)and diluted to obtain 5×10⁴ CFU/mL. 50 μL of this suspension were thenused to inoculate agar plates (20 mL) or culture tubes containing thecorresponding media (930 μL). Test compounds were dissolved in DMSO andadded to the inoculated plates or tubes (20 μL) at concentrationsranging from 1000 to 0.1 μg/mL. The plates and tubes containing cellcultures and compounds were incubated at 270° C. for 72-96 hours. At theend of the incubation period, MIC₁₀₀ (the concentration of the testcompound causing total inhibition of the test organism when compared tothe control) for the test compounds were recorded for each testorganism. Controls were prepared by adding DMSO (20 μL) to theinoculated tube or plate (Roth, G. N., et al., J Nat. Prod. 61 542-545(1998); and Ramsewak, R. S., et al., J. Agric. Food Chem. 47 2, 444-447(1999)). The results are shown in Tables 1 and 2.

TABLE 1 List of samples used for testing zones of inhibition. CODEDESCRIPTION C DMSO Control Ca Camphor M Menthol T Thymol 1 Cedrusatlantica (Cedarwood Atlas) 2 Cedrus deodora (Cedarwood Himalayan) 3Juniperus virginiana (Cedarwood Virginian) 4 Eucalyptus citriodora 5Eucalyptus dives (Eucalyptus Peppermint) 6 Eucalyptus globulus 7Eucalyptus radiata 8 Eucalyptus smithii 9 Eucalyptus staigeriana 10Myristica fragrans (Nutmeg) 11 Pinus pinaster (Turpentine) VE MeijerVaporub - ether extract VM Meijer Vaporub - methanol extract M1 Mixtureof Ca, M, T, and 1-11 (14 samples) M2 Mixture of Ca, M, T, and 4 (4samples)

TABLE 2 Zone of Inhibition (mm) for test samples after 7 days againstorganisms causing toe nail fungus at 250 μg/mL concentrations. X denotesno observable zone of inhibition. VE, VM, M1 and M2 were tested at ahigher concentration, 250 mg/mL. Organism C Ca M T 1 2 3 4 5 6 7 8 9 1011 VE VM M1 M2 Acremonium X 10 15 20 X X 7 14 9 13 11 14 8 6 5 7 14 3737 chrysogenum A. strictum X X 9 12 X X X X X 7 X 8 X X X 3 4 12 25Aspergillus flavus X X X 6 X X X X X X X X X X X 5 7 10 20 A. terreus XX X 3 X X X X X X X X X X X 3 3 7 15 Candida albicans X 7 14 17 X X X 12X 15 12 12 X X X 10 15 20 26 C. kruseii X X 12 16 X X X 9 X 8.5 X X X XX 3 10 11 16 C. parapsilosis X 7 11 20 X X X X X X X X X X X 4 11 14 20Epidermophyton X X 2 X X X 7.5 5 5 2 X X 4 X 3 4 7 38 38 floccosumFusarium X X X 2 X X X X X X X X X X X X X 10 20 oxysporum F.proliferatum X X X 3 X X X 4 X X X X X X X 3 5 12 20 Microsporum canis X2 3 20 X 9 6 11 7 X 6 9 13 X X 2 4 38 38 Scopulariopsis X X X 14 X X X XX X X X X X X X X 4 10 brevicaulis Scytalidium X X X X X X X X X X X X XX X X X 10 15 dimidiatum S. hyalinum X X X 5 X X X 1 X X X X X X X X X12 14 Trichophyton X X X 10 X X X X X X X X X X X X X 38 38mentagrophytes T. rubrum X 8 15 16 X X X X X X X 2 X X X X 4 38 38

EXAMPLE 2

The various test organisms were cultured in Petri dishes containing 20mL of the respective medium (YMG, PDA, Malt Agar, or Sabouraud's agar).The cells from a fully grown plate of each organism were suspended insaline solution (5 mL) and diluted to obtain 5×10⁴ CFU/mL. 1 μL of thissuspension was then used to inoculate 24-well plates containing thecorresponding media and agar (1 mL). Test compounds were dissolved inDMSO, acetone/EtOAc (1:1) or acetone/iso-amyl acetate (1:1) and added tothe inoculated plates (20 μL) at concentrations ranging from 5000 to1000 μg/mL incubated at 27° C. for 7 days. At the end of the incubationperiod, the MIC₁₀₀ (the concentration of the test compound causing totalinhibition of the test organism when compared to the control) for thetest compounds were recorded for each test organism. Controls wereprepared by adding 20 μL DMSO, acetone/EtOAc (1:1) or acetone/iso-amylacetate (1:1) to the inoculated plate.

Procedure 1 for MIC₁₀₀ Determination Using 24-Well Agar Plates at 1000to 250 μg/ml of M2*.

The various test organisms are cultured in Petri dishes containing 20 mLof the respective medium (YMG, PDA, Malt Agar, or Sabouraud's agar).

The cells from a fully grown plate of each organism are suspended insaline solution (5 mL) and diluted to obtain 5×10⁶ CFU/mL.

50 μL of this suspension is then used to inoculate 24-well platescontaining the corresponding media and agar (1 mL).

Test compounds are dissolved in DMSO, acetone/EtOAc (1:1) oracetone/iso-amyl acetate (1:1) and added to the inoculated plates (20μL) at concentrations ranging from 1000 to 250 μg/mL incubated at 27° C.for 7 days.

At the end of the incubation period, the MIC₁₀₀ (the concentration ofthe test compound causing total inhibition of the test organism whencompared to the control) for the test compounds are recorded for eachtest organism.

Controls were prepared by adding 20 μL DMSO, acetone/EtOAc (1:1) oracetone/iso-amy acetate (1:1) to the inoculated plate. *Mixture of Ca,M, T and Eucalyptus citriodora.

Procedure 2 for MIC₁₀₀ Determination using 24-Well Agar Plates—1000 to125 pg/mL of M2.

The various test organisms are cultured in Petri dishes containing 20 mLof the respective medium (YMG PDA, Malt Agar, or Sabouraud's agar).

The cells from a fully grown plate of each organism are suspended insaline solution (5 mL) and diluted to obtain 5×10⁶ CFU/mL.

1 μL of this suspension is then used to inoculate 24-well platescontaining the corresponding media and agar (1 mL).

Test compounds are dissolved in DMSO, acetone/EtOAc (1:1) oracetone/iso-amyl acetate (1:1) and added to the inoculated plates (20μL) at concentrations ranging from 1000 to 125 μg/mL incubated at 27° C.for 7 days.

At the end of the incubation period, the MIC₁₀₀ (the concentration ofthe test compound causing total inhibition of the test organism whencompared to the control) for the test compounds are recorded for eachtest organism.

Controls were prepared by adding 20 μL DMSO, acetone/EtOAc (1:1) oracetone/iso-amyl acetate (1:1) to the inoculated plate.

Procedure 3 for MIC₁₀₀ Determination Using 24-Well Agar Plates at5000-1000 μg/mL of M2.

The various test organisms are cultured in Petri dishes containing 20 mLof the respective medium (YMG, PDA, Malt Agar, or Sabouraud's agar).

The cells from a fully grown plate of each organism are suspended insaline solution (5 mL) and diluted to obtain 5×10⁶ CFU/mL.

1 μL of this suspension is then used to inoculate 24-well platescontaining the corresponding media and agar (1 mL).

Test compounds are dissolved in DMSO, acetone/EtOAc (1:1) oracetone/iso-amyl acetate (1:1) and added to the inoculated plates (20μL) at concentrations ranging from 5000 to 1000 μg/mL incubated at 27°C. for 7 days.

At the end of the incubation period, the MIC₁₀₀ (the concentration ofthe test compound causing total inhibition of the test organism whencompared to the control) for the test compounds are recorded for eachtest organism.

Controls were prepared by adding 20 μL DMSO, acetone/EtOAc (1:1) oracetone/iso-amyl acetate (1:1) to the inoculated plate.

The results are shown in Tables 3 and 4.

TABLE 3 List of samples used in MIC₁₀₀ determinations. CODE DESCRIPTIONC DMSO Control A/E Acetone/Ethyl acetate (1:1) A/I Acetone/Isopentyl(isoamyl) acetate (1:1) Ca Camphor M Menthol T Thymol 4 Eucalyptyscitriodora M2 Mixture of Ca, M, T, and 4 (4 samples) in DMSO M3 Mixtureof Ca, M, T, and 4 (4 samples) in A/E M4 Mixture of Ca, M, T, and 4 (4samples) in A/I

TABLE 4 MIC₁₀₀ (μg/mL) for mixtures M2, M3 and M4 against organismscausing toe nail fungus. Organism M2 M3 M4 Acremonium chrysogenum  7501000 1000 A. strictum 5000 5000 5000 Aspergillus flavus 5000 5000 5000A. terreus 5000 5000 5000 Candida albicans 1000 2000 2000 C. kruseii1000 2000 3000 C. parapsilosis 5000 5000 5000 Epidermophyton floccosum2000 2000 2000 Fusarium oxysporum 3000 4000 4000 F. proliferatum 30004000 4000 Microsporum canis  750 2000 2000 Scopulariopsis brevicaulis5000 5000 5000 Scytalidium dimidiatum 4000 5000 5000 S. hyalinum 50005000 5000 Trichophyton mentagrophytes 3000 4000 4000 T. rubrum 2000 30004000

EXAMPLE 3

The solvents used in the present invention as the topical carrier arepreferably esters of alcohols, such as isoamyl acetate. These carriersare used in nail polish remover, for instance, and provide a fruitysmell to the composition.

The grease or jelly used in the Vapo Rubs available on the market arenot essential and in fact are deleterious since they stain clothing. Thetopical carriers of the present invention are provided in solvents whichevaporate or those which are absorbed into the skin and nails. All ofthis is well known to those skilled in the art.

Over the past decade, faculty in the College of Nursing at MichiganState University, East Lansing, Mich. have been involved in providingbasic foot care services to elderly individuals in the community. Duringthat time a multitude of individuals with varying levels of fungal nailinvolvement have sought foot care services. Initially, over-the-countertopical treatments were suggested as possible remedies. Patients oftenreturned for subsequent foot care and reported that they had not carriedthrough with treatment of over-the-counter anti-fungal medicationsbecause they could not see any improvement.

A “folk remedy” was reported to nurses by one of the individualsreceiving foot care at the clinics. An elderly gentleman reported thathe had totally cleared a bad case of toenail fungus in several months bydaily applying VICKS® VAPO-RUB® (Procter & Gamble, Cincinnati, Ohio) tothe affected nails. Subsequently, nurses suggested trying this treatmentto several other patients at the foot clinics who had obvious toenaildisfigurement due to fungal infection. VICKS® is comprised of thefollowing as active ingredients:

Camphor 4.8%

Menthol 2.6%

Eucalyptus Oil 1.2%

Other ingredients are:

Cederleaf Oil, Nutmeg Oil, Special Petrolatum, Spirits of Turpentine,Thymol.

This original small group of individuals to whom VICKS® nail treatmentwas suggested was followed over the following months. The majorityfollowed through with application of VICKS to their affected nail(s) ona regular or “almost always” basis. They were familiar and comfortablewith the product and, at first, seemed amused that it might have apotential use beyond treating head and chest colds.

The nail changes that were observed were impressive. When individualsreturned two months after they had begun applying VICKS® to fungal nailsthere was a clear line apparent on affected nails demarcating thepresence of fungus from normal colored healthy nail. Over time, as nailgrowth continued, healthy nail replaced that which had been afflictedwith a fungal infection. People continued daily application of VICKS®until all of the fungal nail had grown out and been replaced by healthynail.

The treatment that was suggested to people with fungal infections oftheir toenail included massaging VICKS® into the nail once a day.Bedtime application, combined with wearing white cotton socks to bed toprotect bed linens, was recommended. Orange wood sticks were provided sothat persons who had the manual dexterity to do so could apply Vicks tothe underside of the nail as well as the surface.

Charts were reviewed for 131 individuals who have attended the Collegeof Nursing Foot Care clinics since 1995 when the use of VICKS® was firstsuggested as therapy for toenail fungus. Only active charts werereviewed, so this summary does not include the information from thosepersons which may have had Vicks recommended to them and subsequentlyexpired or have been institutionalized. The college of Nursing foot careclinics are provided at local senior centers and, therefore, themajority of the persons taking advantage of these clinics are elderly.

Sociodemographics:

Seventy percent of the people who have active charts at the foot clinicswere female, a proportion that is consistent with the populationdemographics for persons in the age range who take advantage of theclinics. The majority of clients are quite elderly and range in age from60 to 104. A Table 5 (see also FIG. 1) of the age range of clientsfollows:

TABLE 5 Age Range Percent of Total 60-69 5% 70-79 39% 80-89 42% >90 6%

Additionally, information has been documented relating to the chronicdiseases that clients at the foot clinics report. The following Table 6(see also FIG. 2) indicates the incidence of the five most commonchronic diseases reported by foot care clients.

TABLE 6 Disease Percent of Total Arthritis 44% hypertension 39% CoronaryArtery Disease 20% Diabetes 14% Alzheimer's Disease  5% *It should benoted that percentages do not add up to 100% because some clients doreport multiple chronic diseases.

Of the 131 active charts for our foot care clinics it has beendocumented that 85 of these persons (54%) presented with evidence offungal toenails. VICKS® therapy was recommended for those 85 persons andthe following outcomes were documented. It is important to keep in mindthat these clinics have not been conducted for the purpose of researchand that documentation has been considerably less than systematicrelating to the outcomes resulting from prior recommendation of VICKS®for fungal infection therapy. The results are shown in Table 7 (FIG. 3).

TABLE 7 Number, Percentage of Clients Outcome 19 (22%) Only one visitdocumented 21 (25%) No record of compliance or change  9 (10%) Reportsof non-use 32 (38%) Documentation that fungal infection is cleared

The 25% of clients for whom no record of compliance or change are mostprobably the result of multiple clinicians at the foot care clinics andthe fact that there was no intent, until recently, to document theoutcome of VICKS® therapy. The reasons for non-use of recommended VICKS®therapy by clients include the following (1) wife objects to odor, (2)client cannot reach toes to apply VICKS®, and (3) client cannot rememberto use.

For the 38% of clients for whom fungal infections of their toenails werecleared, the following Table 8 (FIG. 4) summarizes the length of timethat it took before elimination of the infection occurred:

TABLE 8 Length of Treatment Number, Percentage Five Months 3 (10%) SevenMonths 8 (25%) Nine Months 11 (34%)  Eleven Months 4 (12%) >ElevenMonths 6 (19%)

The problem with VICKS® for use in treating nail infections is that thebase is greasy, the active ingredients for fungal nail disease areunknown as are the necessary amounts. There are inactive ingredients inVICKS® which are unnecessary for the treatment of a nail infectioncaused by dermatophytes.

EXAMPLE 4

The goal of this protocol is to vigorously test the effectiveness of theVICKS® treatment in reducing or eliminating toenail fungus. The protocolis also useful for treating the patients with the compositions ofExamples 3 and 4. The basic study is a randomized control group design(Friedman, Furberg & DeMets, Fundamentals of Clinical Trials, 3rd ed.St. Louis, Mo.: Mosby (1996)) with repeated measures of the outcomes.Eligible subjects who agree to participate are divided into threegroups, defined by different levels of exposure to the intervention.

Group I receives VICKS® from the outset (after the intake assessment).Group 2 receives a placebo (of petroleum jelly) for the first month tobe followed by two months of VICKS®, and group 3 receives two months ofplacebo treatment before VICKS® is employed during the last month.Starting at intake, subjects receive, at each monthly visit to theclinic, an unmarked, sealed, dark-colored, 1.5 ounce jar containingeither Vicks Vapo-Rub or the placebo. The content of the jar is unknownto the patient and treating nurse. The intent of this “masking”(Meinert, C. L., Clinical Trials: Design, Conduct, and Analysis. NewYork, N.Y.: Oxford University Press (1986)) is primarily to convincepatients to stick to the prescribed external applications. This is notan example of “complete masking” which would make the odor, quality andtexture of the applications indistinguishable from each other. That isto say, patients will know when they are switched from petroleum jellyto VICKS®. But they do not know which application contains the effectiveingredient. The sealed package also masks the identity of theintervention from the treating nurse creating a “double-blind” design.The purpose here is to minimize bias in measurement.

Subjects are randomly assigned to the three levels of the interventionusing subject recruitment site as an additional blocking variable. (Siteblocking is done to control for likely differences in subjectcomposition by site.)

All measurement and assessments are conducted at intake and at threeadditional monthly follow-up meetings at the foot clinic. A total of 12patient assessments (A_(ij), wherein I=0, 1, 2, 3 refers to the intakeand three follow-up assessments and j=1, 2, 3 refers to the threecomparison groups) will be carried out. The combination of abetween-group design and a repeated measures design allows for testingthe effectiveness of the intervention through multiple, independenthypotheses.

For example, at time 1 after the first month of the intervention, group1 should show a lower assessment means score (=a better toenail outcome)than the two placebo groups: A₁₁<A₁₂=A₁₃. At times 2 or 3, we wouldexpect the following outcomes: A₂₁<A₂₂<23 or A₃₁<A₃₂<A₃₃, since group 1will consistently have longer exposure to VICKS® than group 2, and group2 longer exposure than group 3. Another way of testing the effectivenessof the intervention is by means of within-group comparisons of therepeated measures, i.e., comparisons of assessment scores over time fora given group. Including the baseline pre-test at intake, there are fouravailable measures for each individual. In group 1 where the effectiveintervention is started right away (time 0), we hypothesize thefollowing: A₀₁>A₁₁>A₂₁>A₃₁; in group 2, effective intervention starts attime 2, so we hypothesize: A₀₂=A₁₂>A₂₂>A₃₂. By the same reasoning thehypothesis for group 3 is: A₀₂=A₁₂=A₂₂>A₃₂.

OUTCOME MEASURES

All measurements and assessments were conducted at intake and at threeadditional Assessment of progress towards normal toenails areaccomplished by means of the “Structured Assessment Record Sheet” (seeFIG. 5 for the instrument and the associate instructions for use). Thismeasurement instrument has already been pretested on 14 patients whowere independently assessed by a total of three nurses. With thisinstrument, patients' toenails are assessed on three dimensions: color,thickness and separation from the nail bed. Each toenail is ratedseparately on a rating scale ranging from 0 to 3. The resulting overallnail assessment score can range from zero (all ten toes of both feet arehealthy) to 90 (10 toes×3 rating dimensions×individual maximum scores of3). The distribution of actual scores in the small pilot sample of 28assessments was as follows: 10.5 (means, 8.4 (standard deviation), 0-33(range), 1.6 (skew). Eleven patients were assessed twice by independentnurse raters. The overall assessment scores from the independent raterscorrelated at a level of r=0.915 indicating that the administration ofthis assessment tool will result in a highly reliable measurementprocedure.

In addition to the “objective” toenail assessment by the provider,patient reactions and experiences will also be recorded as well as theircompliance with the therapeutic regimen. We expect “reported change” and“reported pain” to be correlated with the objective assessment. Theadditional subjective, ordinal compliance rating will yield a potentialtool for comparing the compliance records of the three interventiongroups.

SAMPLE SELECTION

A sample of 142 patients who have fungal infections of the toenail arerecruited to take part in empiric testing of VICKS® as a treatment.Subjects are recruited from three sites outside of the area whereprevious foot care clinics have been conducted.

Subjects who are eligible to take part in testing meet the followingcriteria; (1) they have a fungal infection in at least one toenail, (2)they are not pregnant, (3) they are not minors, and (4) they are nottaking oral anti-fungal medications and have not taken any suchmedications within the past three months.

Criteria for identification of fungal infection will include at leastone of the following markers; (1) discoloration of the toenail ascompared with the color of healthy fingernails, (2) thickening of thetoenail, (3) crumbling of the toenail, and (4) accumulation of debrisunder the toenail.

SAMPLE SIZE

For optimal comparisons among the three intervention groups, the randomassignment should yield comparison groups of equal size. Since some ofthe hypotheses involve between-group comparisons and others within groupcomparisons, required sample sizes vary depending on the test. The goalis to obtain data that result in statistically powerful tests of themain hypothesis that VICKS® has therapeutic effects in the treatment oftoenail fungus. The first step in calculations of statistical power isto determine a meaningful “effect size” (Cohen, Jacob, Statistical PowerAnalysis for the Behavioral Sciences, 2nd ed. Hillsdale, N.J.: LawrenceErlbaum Associates (1988), Lipsey, Mark W., Design Sensitivity:Statistical power for Experimental Research. Newbury Park, Calif.: SagePublications (1990)) for our intervention.

A look at the assessment tool devised for this study reveals that theobjective outcome scores are comprised of three subscales (measuring thethree dimensions of color, thickness, and separation). On eachdimension, the minimum score increment is 1. As one might expect, thescores on the assessment dimensions are positively correlated ® >0.5).This means that observable improvement in a single toenail is likely toresult in a score increment of 3. This increment becomes our minimum(absolute) effect size by which we want the comparison groups to differ.

For statistical power calculations, we need an estimate of thestandardized effect size which, for a three-way comparison, can bedefined as ES=σ_(g)/(σ_(i)+σ_(g)), wherein σ_(g)=the standard deviationof the comparison group means and σ_(i) denotes the standards deviationof the remaining individual variation in the population. As can easilybe seen, if the group means do not differ (=the null hypothesis of noeffect) then both σ_(g) and ES will be zero. If there is no individualwithin-group variation but each group mean differs from the others(=perfect effectiveness), then σ_(i) will become zero and ES=1.

In order to translate the absolute effect size into the standardizedeffect size ES, we rely on Cohen's transformation:ES=d/2×(k+1)/(3(k−l))^(½), where d=the standardized difference betweenthe highest and lowest group mean, and k=the number of comparison groups(Cohen, J., Statistical Power Analysis for the Behavioral Sciences. 2nded. Hillsdale, N.J.: Lawrence Erlbaum Associates (1988)). With threecomparison groups, there are two group differences of 3 (as measured inactual outcome scores). Thus, if the two extreme groups in thepopulation differ by more than a score of 6, our test should show astatistically significant finding.

To get the standardized effect, we divide the difference score by theestimated (from the pilot sample) within-group standard deviation of thescores. Thus d=2×3/8.4=0.714. With k=3, our estimate of the standardizedeffect size ES=0.292. In order to be able to discover a real effect sizeof this magnitude 90 percent of the time (=statistical power of 0.90)while maintaining a conventional significance level of 0.05, therequired sample size is 49 subjects in each comparison group (see powertables on p. 314 in Cohen, 1988). It is important to note that thesesample size projections are based on the hypotheses requiring thelargest samples. The repeated measures comparisons of within-groupchanges over time require smaller samples (Lipsey, Mark W., DesignSensitivity: Statistical Power for Experimental Research. Newbury Park,Calif.: Sage Publications (1990)). In addition, gender blocking andcovariate analysis (involving subject age, for instance) may furtherincrease the statistical power of the proposed tests by reducing errorvariance.

In short, we believe that a sample size of 45 cases in each comparisongroup will constitute a powerful test of the main hypothesis with agreater than 90% probability that we will have a statisticallysignificant finding when, in fact, Vicks Vapo-Rub has an effect.

PATIENT RECRUITMENT SITES AND RECRUITMENT TIME TABLE

Elderly patients are recruited from three sites. Sites are located incommunities at least twenty miles from the places where prior foot careclinics have been held to avoid “contamination” related to earlierknowledge or experience with suggested treatment with Vicks. Each sitehas a foot care clinic that takes place once a month in the seniorcenter of a small community. Individuals who take part in the foot careclinics were expected to range in age from 60 to 95 and, based onprevious experience with foot care clinics, will probably bepredominated by women. (60-70% of the individuals who attend currentfoot care clinics are female). Also based on prior experience, we expectover half of the persons who appear at the foot clinics to have fungalinfections of their toenails. Each week, eighteen to twenty individualsare scheduled for foot care at each of the four-hour clinics.

The total observation period for the study was 12 months. Given anintervention and measurement period of 3 months, the phased recruitmentperiod will be limited to a maximum of 9 months. In order to end up witha total of 135 subjects available for analysis (45 in each comparisongroup) and assuming a 5% non-compliance among subjects who initiallyagree to participate, 142 subjects must be enrolled (135/0.95=142) overthe 9-month period. This is equivalent to 15 or 16 patients per monthfrom all three sites combined.

The numbers in the following Table 9 provide an approximate estimate ofthe number of subjects that will be simultaneously enrolled in the study(see bottom line: monthly load). Built into the table is the assumptionthat 7 subjects (or 5%) will be lost due to attrition/non-compliance.

TABLE 9 Phased Enrollment and Observation of Subjects over 12 months.Recruitment (italicized) and Observation Periods: Months since Intake ofFirst Subject 1 2 3 4 5 6 7 8 9 10 11 12 15 15 15 15 16 15 15 15 16 1515 15 16 15 15 15 16 15 15 15 16 15 15 15 16 15 15 15 16 15 15 15 15 1515 15 Monthly 15 31 46 61 61 61 61 61 60 45 30 15 Load

In order to maintain the stratification scheme by location site, monthlyenrollment maintained a fixed proportion of cases from each of the threesites.

RECRUITMENT PROCEDURES

Individuals who attend the foot care clinics are screened for thepresence of toenail fungus during the course of routine foot care. Thosepersons who do have fungal infections of their toenails are informed ofthe study and offered the opportunity to participate. It is explainedthat participation in the study involves daily use of a product that mayor may not be effective in alleviating the fungal infection andrestoring the toenail to a normal, healthy state. Subjects are assuredthat the products to be tested will not cause new damage to the nail andthat they will receive the products free of charge.

Additionally, the subjects agree to refrain from the use of nail polishduring the course of the study and to return to foot care clinics once amonth for assessment of their feet. Routine foot care is providedmonthly at no charge to the subject. All subjects in the threecomparison groups will be provided with identical, written explanationsof the study and will be given instructions for use of the product thathas been provided to them. Each subject is asked to sign a consent formaffirming that the study and the requirements of their participation wasexplained to them and that they had the opportunity to ask questionsabout the study. They are made aware that their participation iscompletely voluntary and that their decision to participate or notparticipate will in no way affect the availability of foot care servicesto them. They are assured that they may withdraw from the study at anytime without penalty.

TRAINING OF NURSE DATA COLLECTORS

Data was collected by a team of four registered nurses who will conductall of the foot care clinics. All nurses who participated in the studywere trained in the provision of basic foot care and in the use of theassessment tool. Practice sessions were conducted with multiple ratersrating the same patient's toenails. Results were compared and assessmentinstructions refined for maximum reliability. A written instructionsheet for use of the assessment tool was provided to each nurse on thefoot care team.

PHASES OF RESEARCH

Phase 1:

During the first three months prior to data collection, the program:hired staff, complete arrangements at the three data collection sites,obtain final approval from institutional review boards, trained nurseinterveners and data collectors and finalize intervention probes andinstruments for data collection.

Phase 2:

During the second nine-month period, all 142 study subjects arerecruited and intake assessments are conducted on all of them.

Phase 3:

This study phase concerns the data collection period for the outcomemeasures. It overlaps with Phase 2, but started one month after theenrollment of the first study subject and ends at the end of the 12thmonth when the last recruited subject completed the intervention andobservation cycle of three months. During this phase, an approximate(depending on attrition) total of 420 patient assessments are processedat their monthly clinic visits.

Phase 4:

This final phase includes data analysis and the writing of the finalreport.

STATISTICAL ANALYSIS PLAN

As outlined in the Study Design Section, the goals of this researchinvolved testing the effectiveness of the VICKS® treatment in reducingor eliminating toenail fungus. The study design is a randomized blockdesign with repeated measures and the outcome measure approximates acontinuous score variable. In sum, the resulting data is likely to meetthe assumptions underlying multivariate analysis of variance orcovariance models (Neter et al., Applied linear statistical models 2nded. Homewood, Ill.: RD Irwin (1985)). The specific hypotheses listedabove was tested against the null-hypothesis that all group and timemeans (A_(ij)) will be equal.

SUPPLIES

A six month supply of Experimental and Control Products in unmarked,coded containers is obtained for each subject in the study. Because thestudy is conducted within the context of foot care clinics it wasnecessary to have foot care supplies in enough quantity to conduct fourclinics each month over the course of a year. Paper supplies will beobtained to record assessments, provide information to subjects, recordinformed consent and maintain study records.

EQUIPMENT

A Polaroid® camera is used to photograph each subjects feet at intakeand following treatment with Vicks Vapo-Ru. Photographs are maintainedwith the subjects records and provide visual evidence of changes in nailhealth.

The procedure set forth above provides a reliable basis for patienttesting of all of the compositions of the present invention. Directionsfor “Structured Assessment Record Sheet” (FIG. 5).

Nail Assessment

Color: Score from “0” for normal to “3” for dark brown/black for eachtoe. The total score for each foot will range from “0” to “15”. Placethe total score for each foot in the coding box for that foot.

Thickness: Score from “0” for normal to “3” for very thick and crumblyfor each toe. The total score for each foot will range from “0” to “15”.Place the total score for each foot in he coding box for that foot.

Separation from Nail Bed: Score from “0” for none to “3”, forsignificant for each toe. The total score for each foot will range from“0” to “15”. Place the total score for each foot in the coding box forthat foot.

PATIENT REACTION

reported Change: Score from “3” for definitely better to “0” for worsefor each foot. The total score for each foot will range from “0” to “3”and should be recorded in the appropriate box for each foot.

Reported Pain: Score from “3” for none to “0” for significant (<5 on ascale of 0-10 where 0=“unbearable” and 10=“none”). The total score foreach foot will range from “0” to “3” and should be recorded in theappropriate box.

Frequency of Treatment: Score from “3” for treatment used daily to “0”for treatment hardly ever used. The total score for each foot will rangefrom “0” to “3” and should be recorded for each foot in the appropriatebox.

It is intended that the foregoing description be only illustrative ofthe present invention and that the present invention be limited only bythe hereinafter appended claims.

We claim:
 1. A method for inhibiting dermatophytes in an infected nailin humans which comprises; multiple separate applications to the nail ofan effective amount of the ingredients camphor, menthol, eucalyptus oil,and thymol in a topical carrier, which is a solvent for the ingredients,is absorbed by the nail and skin, and is not a grease or jelly, untilthe dermatophytes are inhibited, wherein the dermatophytes, which areinhibited by the composition, are Acremonium chrysogenum, A. strictum,Aspergillus flavus, A. terreus, Candida albicans, C. kruseii, C.parapsilosis, Epidermophyton floccosum, Fulsarium oxysporum, F.proliferatum, Microsporum canis, Scopulariopsis brevicaulis, Scytalidiumdimidiatum, S. hyalinum, Trichophyton mentagrophytes, and T. rubrum. 2.The method of claim 1 wherein the composition contains each of theingredients in an amount between 0.01 and 25% by weight of thecomposition.
 3. The method of claim 1 wherein the eucalyptus oil is fromEucalyptus citriodora.
 4. The method of claim 1 wherein the compositioncontains 4.8% camphor, 2.6% menthol, 1.2% eucalyptus oil by weight andan amount between 0.01 to 25% by weight of thymol which is effectiveagainst the dermatophytes.
 5. The method of claim 1 wherein the solventis an ester of an alcohol.
 6. The method of claim 1 wherein the solventis isoamyl acetate.
 7. A method for inhibiting dermatophytes in aninfected nail in humans which comprises multiple separate applicationsof the ingredients camphor, menthol, eucalyptus oil, and thymol in atopical carrier to the nail until the dermatophytes are inhibited,wherein the composition has a minimum inhibitory concentration (MIC₁₀₀)of at least 750 μg/ml of the composition in the topical carrier againstAcremonium chrysogenum, A. strictum, Aspergillus flavus, A. terreus,Candida albicans, C. kruseii, C. parapsilosis, Epidermophyton floccosum,Fulsarium oxysporum, F. proliferatum, Microsporum canis, Scopulariopsisbrevicaulis, Scytalidium dimidiatum, S. hyalinum, Trichophytonmentagrophytes, and T. rubrum.